Project Summary/Abstract Immunotherapy is rapidly becoming a mainstream treatment of cancers, nonetheless, less than 30% of patients benefit from this approach. Thus, there is an urgent need to develop novel immunotherapeutic agents for patients who do not respond to currently available immunotherapies. Our goal is to identify such novel targets by investigating the molecular mechanisms that drive the generation and maintenance of a distinct class of tumor-infiltrating cytotoxic T lymphocytes (CTLs)?tissue-resident memory cells (TRM). We recently performed the largest unbiased survey of over 100 transcriptomes from purified CD8+ CTLs isolated from tumors of treatment-nave patients with early-stage lung cancer and provocatively showed that TRM were key players in mediating robust anti-tumor immune responses (Nature Immunology 2017). We revealed that TRM was selectively enriched in tumors with a high density of tumor-infiltrating lymphocytes (TILs) and displayed enhanced cytotoxicity and proliferation, implying better anti-tumor activity. We also showed that a higher density of TRM cells in tumors predicted better survival outcomes. Given that only a subset of tumors is enriched for TRM, which mounts an effective anti-tumor response, it is crucial to understand the key signals that drive TRM cell generation and maintenance within tumors. We hypothesize that TRM development likely involves several molecular switches (like transcription factors) that regulate migration, tissue retention, survival and response to local milieu (tumor). Here, we propose to directly analyze the transcriptome and epigenome of tumor-infiltrating TRM CTLs to define the molecular pathways governing TRM development by utilizing genomic tools such as single-cell sequencing, ATAC-sequencing and histone ChIP-Seq. We will also functionally validate the role of one such important candidate pathway, i.e., CD39 signaling, in promoting TRM, given that our recent studies show differential high expression of CD39 on TRM-rich TILs. CD39 has been associated with immunesuppression as well as T memory formation, hence the consequences of CD39 expression are unclear. Given that tumor-infiltrating TRM mount clinically beneficial anti-tumor immune responses, we hypothesize that higher expression of CD39 on TRM cells may preferentially protect them from ATP-induced cell death and enable persistence within tumors. We will evaluate the functional role of CD39 in vivo in the generation and maintenance of tumor-infiltrating TRM by testing effects of CD39 abrogation utilizing transplantable and autochthonous mouse tumor models and CD8+ T cell-specific CD39-knockout mice. Furthermore, CD39 knock-down or overexpression studies will be performed in patient-derived tumor- infiltrating TRM cells in vitro and the effects on TRM activation, apoptosis, proliferation, cytotoxicity and cytokine production will be evaluated. In summary, our studies will provide important insights into the molecular signals that promote robust anti-tumor TRM immune response, which may enable immunotherapeutic targeting of critical pathways and design of vaccines that will aid TRM generation.